Abstract This project will combine molecular dynamics (MD) simulations and other types of molecular mechanics calculations with various experimental methods to characterize the biofilms formed by opportunistic pathogens belonging to the Burkholderia cepacia Complex (BCC). Biofilms are communities of microorganisms where cells are embedded in a macromolecular matrix conferring physico-chemical and biological conditions favorable for cell life. Polysaccharides are primary matrix components contributing to biofilm architecture and function through interactions with themselves and with other biofilm constituents. Biofilms also contain low molecular weight compounds having important biological roles, like signaling (e.g. quorum sensing). Polysaccharides may also interact with these molecules, participating in regulatory mechanisms and impeding the effective action of antibiotics. The primary focus of this project will be on the role of the various exopolysaccharides synthesized by the BCC in the formation and maintenance of biofilms. The aim will be to characterize the biofilms on the molecular level and to thoroughly determine polysaccharide structure and conformations, how they control water and adhere to surfaces, and how their properties depend on structural details. Polysaccharide interactions with biofilm constituents, antibiotics, and potential disaggregating agents will also be investigated. The ultimate objective is to obtain information necessary for rational design of therapeutic agents capable of disrupting biofilms without the use of antibiotics, which can promote resistant strains. BCC bacteria synthesize different exopolysaccharides, both under non-biofilm and biofilm culture conditions. Their specific roles in biofilm architecture are still largely unknown. The proposed experimental studies will characterize the fine structure of biofilm polysaccharides of B. multivorans C1576, their interactions with relevant biofilm compounds and selected antibiotics. In addition, phage display methodology will be used to search for polysaccharide-interacting proteins. The data produced by these experimental studies will provide structural information necessary to construct the starting models for the MD simulations, as well as measurements of properties that can be calculated directly from the MD simulations, providing a test of the validity of the simulation results. The extraordinary molecular-level detail available from MD simulations will yield information about features of biofilm architecture and physical properties that cannot be obtained by other means. The effects of antibiotics and the mechanism of norspermidine, which has known biofilm disruption activity, will also be analyzed in MD simulations as a guide in the design of other therapeutic biofilm disrupting agents.